Three dimensional heterogeneously differentiated tissue culture

ABSTRACT

The present invention provides an artificial tissue culture comprising a heterogeneous population of cells of at least two different tissue sections, wherein said tissue sections are in a three dimensional structure, method of generating such a tissue and kits suitable for said method or maintain a three dimensional tissue culture.

The present invention relates to the field of modelling artificialtissue cultures.

Development of the human brain is of primary interest in neuroscience,both due to the nature of its complexity and because defects indevelopment of this unique organ can lead to a variety of devastatingneurological disorders. For example, microcephaly (MCPH), a disordermarked by a severely reduced head and brain size, leads to neurologicaldefects with a poor prognosis for normal brain function (Cox et al.2006).

Several genes have been identified as causative for MCPH (Thornton andWoods. 2009), for example ASPM (Bond et al. 2002) and CDK5RAP2 (Bond etal. 2005), and there is evidence for all of them so far pointing to arole at the centrosome or spindle pole of dividing cells (Megraw et al.2011). In particular, ASPM is the human homolog of the drosophilaabnormal spindle (asp) while CDK5RAP2 is the homolog of centrosomin(cnn), both of which regulate centrosomal and spindle organization.

Heretofore, efforts aimed at teasing out pathogenic mechanisms of MCPHand the roles of these proteins in human brain development have reliedupon mouse models. However, mouse mutants for these genes, includingCDK5RAP2 (Barrera et al. 2010, Lizarraga et al. 2010) and ASPM (Pulverset al. 2010), have failed to recapitulate the severely reduced brainsize seen in human patients with mutations in these genes.

Much of the current knowledge of mammalian brain development has comefrom rodent studies, which have revealed many of the fundamentalmechanisms of mammalian neurogenesis. In rodents as well as humans,brain development begins with expansion of the neuroepithelium togenerate a type of neural progenitor termed radial glia (RG) (Götz andHuttner. 2005). These RGs divide at the apical surface within theventricular zone (VZ) either symmetrically to generate two more RGs orasymmetrically to generate a RG and a more differentiated daughter cell,a neuron or intermediate progenitor. These then migrate outward into thesubventricular zone (SVZ) while the neurons continue migrating throughthe intermediate zone (IZ) to populate specified layers within thecortical plate (CP).

Although the human brain follows these same basic principles duringearly development, there are several key differences from rodents thatallow for the striking expansion in neuronal output seen in humans asdevelopment proceeds (Fietz and Huttner. 2011, Lui et al. 2011). Forexample, the human brain exhibits a large population of a novel stemcell termed outer radial glia (oRG) (Fietz et al. 2010, Hansen et al.2010), which can divide symmetrically and asymmetrically, much like theradial glia in the VZ, to expand the neuronal output. This population isonly present to a very limited degree in rodents, whereas in humans theymake up an entirely separate progenitor layer, termed the outer SVZ(OSVZ). Furthermore, the organization of progenitor zones is markedlymore elaborate in humans, exhibiting a SVZ that is split by an innerfiber layer (IFL) into an inner SVZ (ISVZ) and the OSVZ. Both the IFLand OSVZ are completely absent in mouse.

These differences can explain the difficulties in modeling disorderslike MCPH in rodents, and suggest that these disorders may originatefrom defects in neurodevelopmental processes that cannot be examined inmice. Therefore, methods that recapitulate paradigms of human braindevelopment in vitro have enormous potential.

A variety of culture systems have been described for the derivation ofhuman neurons from pluripotent stem cells. Most of these approaches makeuse of so-called neural rosettes (Wilson and Stice. 2006), which displaycharacteristics of neuroepithelium and can be used to drive theformation of pure populations of specific neuronal subtypes. However,these approaches are limited in their capacity to model many aspects ofhuman brain development as they fail to recapitulate the complexity andheterogeneity seen in vivo.

WO 2011/055855 A1 discloses differentiation of human embryonic stemcells into nerve progenitor cells and cup-like protrusion tissue.

WO 2012/013936 A1 discloses differentiation of neuronal cells andcultures. Stem and progenitor cells are disclosed which form rosettestructures.

EP 2 314 671 A1 discloses cultures derived from human embryonic stemcells.

Wang et al. (2011) describe the identification of radial glia-likeprogenitor cells in mice.

While significant progress has been made in developing in vitro modelsof whole organ development for other systems, such as mammary gland(Kenny et al. 2007), intestine (Sato et al. 2009), and retina (Eiraku etal. 2011, WO 2011/055855 A1), a 3D culture model of the developing brainas a whole has not been established. However, previous studies havepointed to a principle of self-organization for several isolated neuraltissues suggesting an approach may be possible. In particular, Eiraku etal. (2008), US 2011/0091869 A1, have described the formation of dorsalcerebral cortical tissue in three-dimensional culture from pluripotentstem cells. This study reveals the remarkable ability for cerebralcortical tissue to self-organize, and these tissues recapitulated manyaspects of early dorsal cortical development. However, the tissuesgenerated were limited in their identity to dorsal cortex of theforebrain, and while the neurons generated displayed pyramidal subtypeidentities and activity, they failed to form discrete layers withstereotypic inside-out organization. Furthermore, characteristics ofhuman brain development, such as the presence of outer radial glialcells and the unique organization of progenitor zones were not present.

It is therefore a goal of the present invention to provide new tissuemodels based on cell cultures, which represent in vivo tissue behavior.

The present invention relates to an artificial three-dimensionalneuronal tissue culture comprising a heterogenous population of cells ofat least two different progenitor and neuronal differentiation layers,wherein at least one progenitor layer comprises outer radial glia cells.The new neuronal tissue is also referred to as “organoid” or “cerebralorganoids” herein. The cerebral organoids display heterogeneousregionalization of various brain regions as well as development ofcomplex, well-organized cerebral cortex. Furthermore, these tissuesdisplay several characteristics specific to humans, namely the presenceof a substantial outer radial glial population and the organization ofextra cortical subventricular zone layers not present in mouse. Thepresence of outer radial glia cells appears to be one of the mostdistinguishing features, but of course others exist as well. Eiraku etal. (2008) for example describes that in their culture radial glia ofcortical tissues decreased after day 12 and apparently failed to developinto outer radial glia cells, outer radial glia being characterized bytheir position as well as morphology (lack of an apical connection tothe fluid-filled ventricular-like cavity). According to the invention,the outer radial glia cells are preferably in a progenitor layer, inparticular, in a subventricular zone removed from the ventricular zonewhere radial glia reside. Other alternative distinguishing features arefurther described below, e.g. the genetic expression markers.

The invention further provides a method of generating an artificialthree-dimensional neuronal tissue culture comprising a multicellularaggregation of pluripotent stem cells, culturing said multicellularaggregation in neural induction medium, further culturing in a threedimensional matrix, preferably a gel, thereby expanding said cells in amulticellular aggregation, wherein said cells are allowed todifferentiate, and culturing said expanded and optionally differentiatedmulticellular aggregation of cells in a suspension culture. Variousprogenitor and neuron populations could be produced, which displayproper organization and behaviour.

Methods for culturing and differentiating stem cells into neuronal cellsand tissues are known from Eiraku (2008), US 2011/0091869 A1 and WO2011/055855 A1, all incorporated by reference. Methods described thereincan be used in the first step of obtaining the inventive tissue,especially the steps of providing a multicellular aggregation ofpluripotent stem cells and culturing said multicellular aggregation inneural induction medium. During the step of culturing the aggregate, thepluripotent stem cells can be induced to differentiate to neural tissue.For providing a multicellular aggregation, it is e.g. possible toculture pluripotent stem cells from said multicellular aggregates.Contrary to these references, the invention further comprises the stepof culturing the cell aggregates in a three dimensional matrix,preferably a gel, which surprisingly resulted in far more advancedtissue development.

The invention particularly relates to a new method for generating large,complex brain tissues using a 3D in vitro culture system. Individualtissue-like sections of different differentiated cells of the inventiveculture can be in a three dimensionally grown arrangement. The resultingcerebral organoids develop a variety of regional identities organized asdiscrete domains capable of influencing one another, much like the brainas a whole. Furthermore, cerebral cortical regions display anorganization similar to the developing human brain as well as thepresence of a considerable oRG population. Moreover, cerebral corticalneurons mature to form various pyramidal identities and even organize inan inside-out manner reminiscent of cortical layers in vivo. Theorganoid can be used to model neurological diseases, e.g. MCPH. Inparticular, the invention demonstrates utilizing patient-derived iPSCsand shRNA electroporations in these organoids to model pathogenesis ofMCPH, a disorder that has been difficult to model in mice.

The inventive organoids can be obtained from culturing pluripotent stemcells. In principle, the cells may also be totipotent, if ethicalreasons allow.

A “totipotent” cell can differentiate into any cell type in the body,including the germ line following exposure to stimuli like that normallyoccurring in development. Accordingly, a totipotent cell may be definedas a cell being capable of growing, i.e. developing, into an entireorganism.

The cells used in the methods according to the present invention arepreferably not totipotent, but (strictly) pluripotent.

In a particular preferred embodiment, the cells of the present invention(including all further embodiments related thereto), are human cells ornon-human primate cells, pluripotent.

A “pluripotent” stem cell is not able of growing into an entireorganism, but is capable of giving rise to cell types originating fromall three germ layers, i.e., mesoderm, endoderm, and ectoderm, and maybe capable of giving rise to all cell types of an organism. Pluripotencycan be a feature of the cell per see, e.g. in certain stem cells, or itcan be induced artificially. E.g. in a preferred embodiment of theinvention, the pluripotent stem cell is derived from a somatic,multipotent, unipotent or progenitor cell, wherein pluripotency isinduced. Such a cell is referred to as induced pluripotent stem cellherein. The somatic, multipotent, unipotent or progenitor cell can e.g.be used from a patient, which is turned into a pluripotent cell, that issubject to the inventive methods. Such a cell or the resulting tissueculture can be studied for abnormalities, e.g. during tissue culturedevelopment according to the inventive methods. A patient may e.g.suffer from a neurological disorder or cerebral tissue deformity.Characteristics of said disorder or deformity can be reproduced in theinventive organoids and investigated.

A “multipotent” cell is capable of giving rise to at least one cell typefrom each of two or more different organs or tissues of an organism,wherein the said cell types may originate from the same or fromdifferent germ layers, but is not capable of giving rise to all celltypes of an organism.

In contrast, a “unipotent” cell is capable of differentiating to cellsof only one cell lineage.

A “progenitor cell” is a cell that, like a stem cell, has the ability todifferentiate into a specific type of cell, with limited options todifferentiate, with usually only one target cell. A progenitor cell isusually a unipotent cell, it may also be a multipotent cell.

With decreasing differentiation capabilities, stem cells differentiatein the following order: totipotent, pluripotent, multipotent, unipotent.During development of the inventive organoid, stem cells differentiatefrom pluripotent (also totipotent cells are possible) into multipotentneural stem cells, further into unipotent stem cells of a cerebral layerand subsequently into non-stem tissue cells. Tissue cells may e.g. beneuronal cells or neuroepithelial cells, such as glial cells.

The inventive tissue culture is in vitro grown, i.e. it is not anisolated brain from an animal during any stages. Since it is grown fromhuman pluripotent stem cells, this allows growth of human brain tissuewithout the need to obtain human fetal brain tissue samples. Inaddition, this system represents growth of derived brain tissue in 3D,whereas isolated animal brain tissues have only been used in 3D togenerate neurospheres, an aggregation of dissociated neural stem cellswith limited multipotent capacity (Reynolds and Weiss. 1992). Theseneurospheres fail to recapitulate many aspects of in vivo braindevelopment e.g. regional identities, progenitor and differentiationlayer organization, neuronal layering organization, which can beprovided by the inventive tissue culture and/or methods. The inventivetissue culture is not and differs according to these aspects from aneurosphere.

During the development, the cell aggregates form polarizedneuroepithelial structures and a neuroepithelial sheet, which willdevelop several round clusters (rosettes). These steps can be controlledby neural induction medium as described by Eiraku (2008), US2011/0091869 A1 and WO 2011/055855 A1. In the absence of neuralinduction medium, e.g. by using standard differentiation media, theinvention further comprises culturing in a three dimensional matrix,preferably a gel, especially a rigid stable gel, which results infurther expansion of neuroepithelium and differentiation. A suitablethree dimensional matrix may comprise collagen. More preferably thethree dimensional matrix comprises extracellular matrix from theEngelbreth-Holm-Swarm tumor or any component thereof such as laminin,collagen, preferably type 4 collagen, entactin, and optionally furtherheparan-sulfated proteoglycan or any combination thereof. Such a matrixis Matrigel. Matrigel is known in the art (U.S. Pat. No. 4,829,000) andhas been used to model 3D heart tissue previously (WO 01/55297 A2).Preferably the matrix comprises a concentration of at least 3.7 mg/mlcontaining in parts by weight about 60-85% laminin, 5-30% collagen IV,optionally 1-10% nidogen, optionally 1-10% heparan sulfate proteoglycanand 1-10% entactin. Matrigel's solid components usually compriseapproximately 60% laminin, 30% collagen IV, and 8% entactin. Entactin isa bridging molecule that interacts with laminin and collagen. The threedimensional matrix may further comprise growth factors, such as any oneof EGF (epidermal growth factor), FGF (fibroblast growth factor), NGF,PDGF, IGF (insulin-like growth factor), especially IGF-1, TGF-β, tissueplasminogen activator. The three dimensional matrix may also be free ofany of these growth factors.

In general, the three dimensional matrix is a three dimensionalstructure of a biocompatible matrix. It preferably comprises collagen,gelatin, chitosan, hyaluronan, methylcellulose, laminin and/or alginate.The matrix may be a gel, in particular a hydrogel. Organo-chemicalhydrogels may comprise polyvinyl alcohol, sodium polyacrylate, acrylatepolymers and copolymers with an abundance of hydrophilic groups.Hydrogels comprise a network of polymer chains that are hydrophilic,sometimes found as a colloidal gel in which water is the dispersionmedium. Hydrogels are highly absorbent (they can contain over 99% water)natural or synthetic polymers. Hydrogels also possess a degree offlexibility very similar to natural tissue, due to their significantwater content.

After the expansion, the cell aggregates can be cultured in suspensionculture, preferably a bioreactor. Said culturing in suspension cultureis preferably also in the absence of neural induction medium. A suitablemedium is a standard differentiation medium.

In preferred embodiment the medium can comprise or lack the followingcomponents:

Medium A for the step of culturing pluripotent stem cells as anaggregate (termed an embryoid body): serum replacement formulation,fetal bovine serum, glutamine, non-essential amino acids,2-mercaptoethanol, bFGF, preferably about 4 ng/ml bFGF, or anycombination thereof. Especially preferred, this medium contains a ROCKinhibitor for the initial stages of aggregate culture. Such a medium ise.g. hES medium used in the examples.

Medium B the step of differentiating the aggregate of pluripotent stemcells to neural tissue: N2 supplement (Price and Brewer. 2001),glutamine, non-essential amino acids, heparin, or any combinationthereof. This medium preferably lacks growth factors that woulddifferentiate neural tissue to a particular fate. Such absent growthfactors may be any one of Shh, Wnt, Bmp, retinoids, or FGF, or anycombination thereof, especially all of them. Such a medium is e.g.neural induction medium used in the examples.

Medium C for the step of culturing in a three dimensional matrix,preferably a gel: N2 supplement (Price and Brewer. 2001), B27 supplement(Price and Brewer. 2001), insulin, 2-mercaptoethanol, glutamine,non-essential amino acids, or any combination thereof. This mediumpreferably lacks growth factors that would differentiate neural tissueto a particular fate. Such absent growth factors may be any one of Shh,Wnt, Bmp, retinoids, or FGF, or any combination thereof, especially allof them. Such a medium is e.g. differentiation medium used in theexamples.

Medium D for the step of culturing in a suspension culture, preferably abioreactor: N2 supplement, B27 supplement, insulin, 2-mercaptoethanol,glutamine, non-essential amino acids, or any combination thereof. Thismedium preferably lacks growth factors that would differentiate neuraltissue to a particular fate. Such absent growth factors may be any oneof Shh, Wnt, Bmp, or FGF, or any combination thereof, especially all ofthem. Preferably this medium contains retinoic acid to promote pyramidaldifferentiation and maturation. Such a medium is e.g. “differentiationmedium+RA” used in the examples.

Any medium further contains nutrients, buffers, oxygen. The medium mayfurther comprise growth factors or lack growth factors. Growth factorswhich may be present or missing are e.g. EGF, FGF, NGF, PDGF, IGF,especially IGF-1, TGF-β, tissue plasminogen activator. Preferrednutrients include a carbohydrate, especially a mono-hexose ormono-pentose, such as glucose or fructose. In preferred embodiments anyone of the media, preferably all, are serum-free.

The step of culturing pluripotent stem cells is preferably performed fora duration of 2 to 8 days, especially preferred 5 to 7 days. Inparticular, said step may be performed on culture days 0 to 8. The stepof culturing the aggregate of pluripotent stem cells is preferablyperformed for a duration of 2 to 7 days, especially preferred 4 to 6days. In particular, said step may be performed on culture days 5 to 14.The step of culturing in a three dimensional matrix, preferably a gel ispreferably performed for a duration of 1 to 6 days, especially preferred3 to 5 days. In particular, said step may be performed on culture days 9to 18. The following step of culturing in a suspension culture ispreferably performed for a duration of at least 3 days, especiallypreferred at least at least 4 or at least 5 days.

In preferred embodiments the suspension culture (especially thesuspension culture after culturing in a 3D matrix) is a stirring orshaking medium culture, in particular preferred a bioreactor. At thisstage, the inventive culture has reached enlarged size dependent onconstant nutrient supply. This is best achieved by flushing of thecells, e.g. by stirring or shaking.

In preferred embodiment, during cell expansion, especially in the 3Dmatrix the cells are allowed to differentiate into unipotent stem cells(progenitor cells). During this step tissue-like development proceedscomprising the formation of distinctive layers, including layers ofunipotent cells occurs, which give rise to specialized nerve orepithelial cells.

The present invention also relates to a cell or tissue cultureobtainable by said methods. In particular, the invention provides an invitro grown artificial three-dimensional neuronal tissue culture(“organoid”) comprising a heterogeneous population of cells of at leasttwo different neuronal differentiation layers. As mentioned above,preferably at least one differentiation layer comprises outer radialglia cell.

The inventive culture may develop into a differentiated tissuecomprising layers of different differentiation grade. In a 3D structurethis may be observable as separate sections of the cultures. Inpreferred embodiments, the culture comprises tissue sections form atleast two layers. Such a layer may be shaped around a globular tissuebody, e.g. a body from which the distinct layer(s) have developed. Inparticular, the tissue may show a distinctive development of apical anddorsal tissue sections.

The inventive tissue is or resembles cerebral tissue comprisingsubstantially all cells found in the brain or progenitors thereof. Suchcells can be identified by selective gene expression markers, which areon a level above the average of not differentiated cells, in particularincluding confidence intervals. Such markers can be identified byspecific oligonucleotide probes, which preferably bind exclusively tosaid target marker nucleic acid, especially target marker mRNA. Markerscan further be identified by specific antibodies.

Preferably cells of the inventive culture express one or more geneexpression markers selected from forebrain markers BF1 and Six3.Alternatively, or in addition, preferably cells of the inventive cultureexpress one or more gene expression markers selected from hindbrainmarkers Krox20 and Ils1. At a certain stage of development forebrainmarkers are expressed in increased amounts as compared to hindbrainmarkers in the tissue. This is preferably reflected in the culture ofthe invention.

The inventive tissue culture can alternatively or in addition becharacterized by comprising cells expressing one or more gene expressionmarkers selected from Otx1, Otx2, FoxG1, Auts2, Tbr2, Tuj1, Brn2, Satb2,Ctip2, calretinin, or any combination thereof. These markers may beexpressed during any stage of the culture during the inventive method,and are preferably expressed in the provided tissue culture.

Preferably the inventive culture comprises cells, which express Otx1and/or Oxt2. Otx1 and/or Oxt2 are expressed in cells offorebrain/midbrain identity. Preferably this tissue type is comprised inthe inventive culture.

Preferably the inventive culture comprises cells, which express FoxG1.FoxG1 is expressed in cells of dorsal cortex identity. Preferably thistissue type is comprised in the inventive culture.

Preferably the inventive culture comprises cells, which express Auts2.Auts2 is expressed in cells of frontal cortex identity. Preferably thistissue type is comprised in the inventive culture.

Preferably the inventive culture comprises cells, which express Tuj1.Tuj1 is expressed in cells of a cortical inner fiber layer identity.Preferably this tissue type is comprised in the inventive culture.Generation of an inner fiber layer (and also an outer subventricularzone) have never been achieved before and are indicators of theinventive tissue.

Preferably the inventive culture comprises cells, which express Brn2.Brn2 is expressed in cells of a later born neuron (neuron of outerregion). Preferably this tissue type is comprised in the inventiveculture.

Preferably the inventive culture comprises cells, which express Satb2.Satb2 is expressed in cells of a later born neuron (neuron of outerregion). Preferably this tissue type is comprised in the inventiveculture.

Preferably the inventive culture comprises cells, which express Ctip2.Ctip2 is expressed in cells of earlier born neuron (neuron of innerregion). Preferably this tissue type is comprised in the inventiveculture.

Preferably the inventive culture comprises cells, which expresscalretinin. Calretinin is expressed in cells of cortical interneuronswithin the dorsal cortical plate. Preferably this tissue type and/or thecortical interneurons is/are comprised in the inventive culture.

The inventive artificial tissue can also be used as a research tool tostudy the effects of any external (e.g. drugs or other stimuli) orinternal (mutations) influences on growth and activity of cells in thetissue. Therefore, in an additional aspect, the invention provides amethod of investigating a developmental neurological tissue effect, e.g.a defect, in particular a developmental defect, comprising decreasing orincreasing the expression in a gene of interest in a cell at any stageduring the inventive method. A gene of interest can be a gene, that issuspected to be essential or detrimental when active during thedevelopment healthy neuronal tissue. Methods to decrease or increaseexpression in a gene are well known in the art, and include knock-out orknock-down methods (especially RNA interference, antisense inhibition,shRNA silencing, etc.), or introductions of transgenes (e.g. knock-in),respectively. Such decrease or increases can be conditional, e.g. byintroducing a genetic construct with inducible promoters and/orconditional knock-out or knock-downs or knock-ins. The introduction ofconditional mutations of essential genes or introductions of lethalgenes are possible by using suitable conditional mutation vectors, e.g.comprising a reversible gene trap. Conditional mutations preferablyfacilitate reversible mutations, which can be reversed to a gene-activeor inactive, respectively, state upon stimulation, e.g. as in thedouble-Flex system (WO 2006/056615 A1; WO 2006/056617 A1; WO 2002/88353A2; WO 2001/29208 A1). Mutations can either be random or site-directedat specific genes. Thus in preferred embodiments of the invention,reversible mutations are introduced into the pluripotent stem cells,either by random (forward) or site directed (reverse) mutagenesis.Suitable vectors comprising insertion cassette with a reversiblemutations. Mutations can be switched on or off at any stage of theinventive method. Vectors or other nucleic acids can be introduced intocells with any method known in the art, e.g. electroporation. It is ofcourse also possible to provide cells having a given mutation. Suchcells can be isolated from a patient, followed by a step of inducingpluripotent stem cell status, and letting the cells develop into theinventive tissue, e.g. by the method described above. The patient mayhave a particular disease of interest, especially a neurological defector cerebral deformity. Such a method has been shown in the examplesbelow for cells of a patient with microcephaly. Genetic mutations ofmicrocephaly, such as a mutation in the gene Cdk5Rap2 leading todecreased expression, are example mutations, which can be investigatedby the inventive method.

The present invention further provides a method of screening a candidatetherapeutic agent suitable for treating a developmental neurologicaltissue defect of interest, comprising performing the above method forinvestigating a mutation and administering the candidate agent to saidcells at any stage during the method, preferably at all stages.According to this aspect, a candidate therapeutic drug can be screenedfor having an effect on any cell with a mutation, which can beintroduced as described above. It is of course also possible to usecells of patients with a given mutation, inducing pluripotent stem cellstatus and performing the inventive methods to induce tissue developmentas described above. In particular, the present invention providesinvestigations in mutations in microcephaly and allows the screening ofpharmaceutical agents, which can affect the mutations, e.g. compensatefor the insufficiency or overexpression in the mutated gene, e.g.Cdk5Rap2 in microcephaly. A positive candidate drug could be a compound,which restores normal cellular development, as can be observed byperforming the inventive tissue generation method without a mutation forcomparison, e.g. by using healthy pluripotent stem cells.

Of course, it is also possible to screen candidate drugs, e.g. candidatetherapeutic drugs, to have any effect on normal tissue as well, withouta mutation, which leads to an aberrant development. Thus in yet anotheraspect, the invention relates to a method of testing a candidate drugfor neurological effects, comprising administering a candidate drug toan artificial culture and determining an activity of interest of thecells of said culture and comparing said activity to an activity ofcells to the culture without administering said candidate drug, whereina differential activity indicates a neurological effect. Any kind ofactivity of the inventive cells or tissue, including metabolic turn-overor neuronal signalling can be searched for in a candidate drug. Inessence, the inventive highly differentiated tissue can be used as amodel for cerebral behaviour testing on any effects of any drug. Such amethod might also be used to test therapeutic drugs, intended fortreating any kind of diseases, for having side-effects on nerves, inparticular brain tissue, as can be observed in the inventive tissueculture.

The present invention can also be used to obtain neuronal cells. Inparticular, the invention provides a method of obtaining adifferentiated neural cell comprising the step of providing anartificial culture and isolating a differentiated neural cell ofinterest, or comprising the step of generating an artificial tissueculture according to the invention further comprising the step ofisolating a differentiated neural cell of interest. Such cells isolatedfrom the inventive culture or tissue have the benefit of representingsimilar morphological properties as cells isolated from cerebral tissueof an non-human animal, as mentioned above, or a human.

The present invention further provides a kit for generating theinventive tissue culture comprising containers with any one of theculturing media described above, especially a medium containing a threedimensional matrix as described above and nutrients and a mediumcomprising retinoic acid and nutrients, optionally further comprising amedium comprising nutrients and a ROCK inhibitor and/or optionallycomprising a medium comprising nutrients and lacking growth factors thatwould differentiate neural tissue to a particular fate.

The kit further comprises a medium C comprising a three dimensionalmatrix, and preferably lacking growth factors that would differentiateneural tissue to a particular fate. Such absent growth factors may beany one of Shh, Wnt, Bmp, retinoids, or FGF, or any combination thereof,especially all of them. This medium preferably further comprises cellnutrients. Especially preferred, the medium comprises N2 supplement(Price and Brewer. 2001), B27 supplement (Price and Brewer. 2001),insulin, 2-mercaptoethanol, glutamine, non-essential amino acids, or anycombination thereof.

The kit further comprises a medium D comprising retinoic acid andnutrients. This medium preferably lacks the three dimensional matrix.Especially preferred, the medium comprises N2 supplement, B27supplement, insulin, 2-mercaptoethanol, glutamine, non-essential aminoacids, or any combination thereof. This medium preferably lacks growthfactors that would differentiate neural tissue to a particular fate.Such absent growth factors may be any one of Shh, Wnt, Bmp, or FGF, orany combination thereof, especially all of them.

Optionally, the kit may further comprises a medium A comprising a ROCKinhibitor and nutrients. Especially preferred, the medium comprisesserum replacement formulation, fetal bovine serum, glutamine,non-essential amino acids, 2-mercaptoethanol, bFGF, preferably about 4ng/ml bFGF, or any combination thereof.

Optionally, the kit may further comprise medium B comprising nutrientsand lacking growth factors that would differentiate neural tissue to aparticular fate. Such absent growth factors may be any one of Shh, Wnt,Bmp, retinoids, or FGF, or any combination thereof, especially all ofthem. Especially preferred, the medium comprises N2 supplement (Priceand Brewer. 2001), glutamine, non-essential amino acids, heparin, or anycombination thereof.

The inventive kit preferably comprises a medium for any one of the stepsdescribed above, selected from the step of culturing pluripotent stemcells, the step of culturing the aggregate of pluripotent stem cells,the step of culturing in a three dimensional matrix, preferably a gel,the step of in a suspension culture, preferably a bioreactor. Inparticular preferred this the combination of a medium for the step ofculturing in a three dimensional matrix, preferably a gel, and the stepof in a suspension culture; or a combination of a medium for the stepsof the step of culturing the aggregate of pluripotent stem cells, thestep of culturing in a three dimensional matrix, preferably a gel.Preferably, the media for performing separate steps are provided inseparate containers, such as vials or flasks. Any one of the inventivemedium may comprise further auxiliary substances such as buffers,stabilizers, nutrients, as mentioned above. The medium may be providedin a solid, dry form or in aqueous form.

It is contemplated that any method or product described herein can beimplemented with respect to any other method or product described hereinand that different embodiments may be combined.

The claims originally filed are contemplated to cover claims that aremultiply dependent on any filed claim or combination of filed claims.

The use of the word “a” or “an” when used in conjunction with the term“comprising” in the claims and/or the specification may mean “one”, butit is also consistent with the meaning of “one or more”, “at least one”,and “one or more than one”.

It is contemplated that any embodiment discussed herein can beimplemented with respect to any method or product of the invention, andvice versa. Any embodiment discussed with respect to a particularcondition can be applied or implemented with respect to a differentcondition. Furthermore, compositions and kits of the invention can beused to achieve methods of the invention.

Throughout this application, the term “about” may be used to indicatethat a value includes the standard deviation of error for the device ormethod being employed to determine the value or in a set value may referto ±10%.

The present invention is further illustrated by the following figuresand examples, without being restricted to these embodiments of theinvention.

FIGURES

FIG. 1. Description and characterization of the cerebral organoidculture system. a. Schematic of the culture system described in moredetail in Methods. Human pluripotent stem cells (hPSCs) were dissociatedfrom colony culture on feeders and transferred to floating aggregatestermed embryoid bodies which begin differentiating into the three germlayers. These were allowed to grow for 6 days in media containing lowbFGF, and then transferred to low adhesion plates containing a definedneural induction media to support neuroectoderm growth while limitinggrowth of other germ layers. On day 11, neuroectoderm tissues weretransferred to Matrigel droplets and grown in floating culture indifferentiation media followed by culture in a spinning bioreactor indifferentiation media containing retinoic acid (RA). Example images ofeach stage are shown below the schematic. b. Neuroepithelial tissuesgenerated using this approach (left panel) were larger and morecontinuous than when grown in stationary suspension without Matrigel(right panel). This approach also generated tissues with larger fluidfilled cavities as well as typical apical localization of the neuralN-cadherin protein (arrow). c. Sectioning and immunohistochemistryrevealed that advanced tissues displayed complex morphology withheterogeneous regions of neural tissues containing neural progenitors(Sox2, red) and neurons (Tuj1, green) (arrow). d. Low magnificationbright field imaging further revealed large fluid-filled cavitiesreminiscent of ventricles (white arrow) as well as a variety ofdeveloping neural tissues including retina, as indicated by the presenceof a retinal pigmented epithelium (black arrow). e. Hemotoxylin-eosinstaining of cerebral organoids compared with stationary culture revealsoverall larger tissues with substructure reminiscent of brain regionssuch as forebrain cortex (arrows) and choroid plexus (arrowhead).

FIG. 2. Human cerebral organoids recapitulate various brain regionidentities. a. RT-PCR for forebrain markers (BF1 and Six3) as well ashindbrain markers (Krox20 and Isl1) in cortical organoids at 12, 16 and20 days of differentiation. Human fetal brain cDNA was used as apositive control. b. Immunohistochemistry for the forebrain/midbrainmarkers Otx1/2 (green) and the hindbrain marker Gbx2 (red) at 16 and 20days of differentiation revealing primarily fore/midbrain identity withadjacent regions of hindbrain reminiscent of the mid-hindbrain boundary(arrows). DAPI marks nuclei (blue). c. Immunohistochemistry for themarker FoxG1 (red) revealing a discrete region of dorsal cortex withinthe organoid. d. Staining for the marker of frontal lobe Auts2 (red)revealing subregionalization of cerebral cortical lobes within theorganoid. e. Staining for Nkx2.1 (red), a marker of ventral corticalidentity, and Pax6 (green) marking dorsal cortex reveals adjacent dorsaland ventral regions. Staining for Calretinin (green) in a serial sectionreveals the production of cortical interneurons in the ventral region ofthe organoid. f. Staining for Neuropilin-2 (Nrp2, red) as well ascostaining of Frizzled-9 (red) and Prox1 (green) revealing hippocampalregions within independent cerebral organoids. g. Immunohistochemicalstaining for Transthyretin (TTR) a marker of choroid plexus, revealingregions which also display typical morphology of the choroid plexus.

FIG. 3. Stereotypical organization of progenitor zones in dorsal cortexof cerebral organoids. a. Immunohistochemistry for neurons (Tuj1, green)and radial glial progenitors (Pax6, red) in a typical large (approx. 1mm across) dorsal cortical region within a cerebral organoid thatrecapitulates the apical-basal organization of progenitors adjacent tothe fluid-filled cavity in a region reminiscent of ventricular zone andnewborn neurons accumulating basally. b. Staining for the IP marker Tbr2(red) revealing a subventricular zone localization much like in vivo. c.Staining for phospho-histone H3 (PH3, green) to mark cells in mitosis.Progenitor divisions primarily occurred at the apical surface, butseveral divisions can be seen is a subventrical region, likely belongingto IPs or oRGs. Pax6 (red) marks radial glia. d. Immunohistochemistryfor phospho-Vimentin (green), a marker of mitotic radial glia revealingtypical division at the apical surface. e. Higher magnification image ofphospho-Vimentin staining (green) of a dividing readial glia revealingthe long basal process typical of radial glial morphology. f. Schematicof electroporation technique. Plasmid DNA was injected into fluid-filledcavities within the organoid and an electric pulse was applied toelectroporate cells (radial glial progenitors) adjacent to the cavity.These results in several regions of electroporation (right panel, GFP ingreen) and high efficiency of electroporation of RGs (lower panel, GFPin green). g. GFP electroporated progenitors (arrows) in an early stagetissue (18 days) revealing neuroepithelial morphology. h. GFPelectroporated tissue at 30 days revealing radial glia (arrows) withtypical bipolar morphology (arrowheads). i. GFP electroporated tissue at36 days revealing more advanced thicker cortical region with radial glia(arrow) exhibiting long apical and basal processes (arrowheads).

FIG. 4. Radial glia of cerebral organoids exhibit typicalcharacteristics seen in vivo. a. Frames from live imaging of anelectroporated radial glia (GFP, green) showing movement of the cellbody (arrow) along the bipolar processes. Time in hours and minutes isshown in upper right. b. BrdU pulse-chase experiment revealinginterkinetic nuclear migration. At 1 hour of BrdU administration, BrdUpositive (green) radial glia (Sox2, red) were located in the basalregion of the VZ. 4 hours after washing out BrdU, many BrdU+ cells canbe seen shifted apically, while at 6 hours after washing, several cellscan be seen at the apical surface. c. Phospho-Vimentin (green) stainingrevealing a mitotic cell at the apical surface during anaphase (arrow)with a planar orientation of division. d. Quantification of radial glialorientation of division relative to the apical surface, displayed inbins of 0-30 degrees (planar), 30-60 degrees (oblique) and 60-90 degrees(verticle). n=27 cells from 5 different cerebral cortical regions. e.Lineage tracing in GFP electroporated tissues following a short one hourpulse of BrdU followed by a 16-hour chase. Daughter cell pairs aremarked by colabeling with GFP and BrdU. Symmetric divisions withdaughter cells of the same identity (Sox2 positive, blue, arrowheads) aswell as asymmetric divisions (arrows) can be observed. f. Quantificationof results shown in e. for 18 cell pairs from three independent corticaltissues. Numbers above bars represent number of daughter pairs for eachcategory.

FIG. 5. Cerebral organoids produce oRGs and neurons with typicalmorphology and migration behavior. a. Staining for Sox2 (red, radialglia) and Tuj1 (green, neurons and processes) reveals the presence ofouter radial glia separated from the apical ventricular zone (VZ) andorganized similar to human cortical development. The VZ and SVZ appearseparated from a layer of oRGs (OSVZ) by a layer of Tuj1+ fibers muchlike the inner fiber layer (IFL). b. Immunohistochemistry forphospho-Vimentin (green) revealing dividing oRGs (arrows) with typicalcell morphology, namely the presence of a basal process (arrowheads) butlacking an apical process. Just after division a daughter cell pair canbe seen, one of which inherits the basal process. Apical (A) is orienteddown while basal (B) is oriented up. c. Staining for phospho-Vimentin(green) in a recently divided daughter cell pair reveals one daughtermaintained as an oRG (Sox2+, red) while the other lacks Sox2 expression(arrowhead). d. Orientation of division of a mitotic oRG in anaphaserevealing vertical (60-90 degrees) orientation relative to the apicalsurface (dashed line). Quantification of this orientation is shown onthe right. e. Immunohistochemistry for the early born neuron markerCtip2 (green) and later born neuron marker Brn2 (red) revealsindependent neuron populations exhibiting rudimentary separation at 30days of differentiation. f. At 75 days of differentiation, separation ofearly born (Ctip2, green) and late born (Satb2, red) is more evidentwith inside-out organization reminiscent of that seen in vivo. g.Calretinin staining (green) for cortical interneurons generated fromventral cortex (FIG. 2 e) exhibit typical morphology of tangentialmigration into the dorsal cortical tissue (FoxG1, red) with leadingprocesses perpendicular to the apical “ventricular” surface. h. GFP(green) electroporated cortical neurons (arrows) extend long-range axonswith evidence of axon bundling (arrowheads) similar to that seen inpyramidal tracts. i. High magnification image of GFP (green)electroporated neural axon displaying complex morphology and axonbranching (arrowheads). j. False color heat map frames from live imagingwith Fluo-4 calcium sensitive dye revealing spontaneous calcium surgesin individual neurons (arrowheads) of cerebral organoid. Time isdisplayed in minutes:seconds.

FIG. 6. Cerebral organoids generated from a patient derived iPSCs orshRNA electroporation model microcephaly a. MRI scan from patient A3842taken at birth (top) compared with age-matched control (bottom) showingbrain and head size reduction and simplified cortical folding (arrows).Saggital T1 (left) and axial T2 (right) images. Scale bar 1 cm. b.Sequencing chromatograms demonstrating compound heterozygous nonsensemutations inherited from each parent. c. Western blot for Cdk5Rap2protein in lysates from control and patient (A3842) skin fibroblastsrevealing loss of the protein in A3842 patient. Vinculin (VCL) is shownas a loading control. d. Immunocytochemical staining for Cdk5Rap2 inpatient (A3842) and control fibroblasts revealing localization tocentrosomes (CPAP, green) in control but lack of staining in patientfibroblasts. e. Representative bright-field images of cerebral organoidsgenerated from control iPSCs and patient derived (line 1M is shown here,all lines are shown in FIG. 9) at 6, 11, 15, and 22 days ofdifferentiation. Control exhibits large fluid-filled cortical regions,while patient derived tissue exhibits increased outgrowth with fewerregions of thick cortical tissue. f. Immunohistochemistry in Control andpatient derived (Line 10M is shown as a representative example) tissuesat day 30 of differentiation revealing fewer neurons (Doublecortin, DCX,green, arrows) and smaller progenitor zones (Sox2, red, arrowheads). g.Staining at an earlier stage (day 22) for neurons (Tuj1, green) andradial glia (Sox2, red) revealing smaller progenitor zones and increasedneurons in patient derived tissues (Lines 1M and 14B are shown here). h.Higher magnification of developing cortical tissues showing increasedneurons (Tuj1, green, arrows) in patient derived (line 14B) tissue. i.hES cell derived organoids co-electroporated with GFP (green) and shRNAsagainst Cdk5Rap2 or a scrambled shRNA. Regions electroporated withCdk5Rap2 shRNAs exhibit loss of Sox2+(red) progenitors and increaseddoublecortin (DCX, blue) neurons. j. Higher magnification of results ini. showing neuronal morphology of GFP (green) electroporated withCdk5Rap2 shRNA. These exhibit increased DCX (blue) expression and a lossof Sox2 (red) compared with scrambled or adjacent non-electroporatedtissue.

FIG. 7. Generation of cerebral organoids from multiple human pluripotentstem cells. a. Hemotoxylin-eosin staining of organoids generated fromhuman H9 ES cells as well as human iPS cells display similar size andcomplex morphology as well as the presence of advanced forebraintissues, shown at higher magnification in the lower panels. b. Stainingfor N-cadherin (green) and newborn neurons (Doublecortin, DCX, red) intissues generated from both human H9 ES cells and human iPS cellsreveals similar organization and in tact apical basal polarity in bothtypes of tissues.

FIG. 8. Neural identity during differentiation of cerebral organoids.RT-PCR of the pluripotency markers Oct4 and Nanog as well as neuralidentity markers Sox1 and Pax6 in undifferentiation human ES cells andfollowing differentiation at 6 and 9 days revealing decreasedpluripotent identity at 9 days of differentiation whereas neuralidentity was activated.

FIG. 9. Characterization of patient derived iPSCs and cerebralorganoids. a. iPS cells derived from A3842 patient skin fibroblastsexhibit typical ES cell-like morphology. Four lines were chosen foranalysis based on this typical morphology and pluripotency. b. Alkalinephosphatase staining (blue) of patient derived iPS cell coloniesrevealing pluripotency. c. Representative early organoid culture ofpatient (line 1M) and control using the protocol and timing establishedfor normal hES cells. Patient tissues were much smaller and failed tothrive so the protocol had to be slightly modified to produce neuraltissues. d. Patient derived tissues using increased starting cell numberdisplay neuroepithelium but do not form thick fluid-filled corticaltissues as seen in control derived tissues. e. Western blot forendogenous Cdk5Rap2 in 293T cells transfected with 4 different shRNAsagainst Cdk5Rap2. shRNA1 and 2 are most efficient while shRNA 4 leads toa modest reduction in protein. Tubulin is shown as a loading control.

FIG. 10. Human cerebral organoids recapitulate various brain regionidentities. a. Staining for the preplate marker Tbr1 (red) and neuronalmarker MAP2 (green) revealing superficial preplate (upper bracket) andunderlying neuronal IZ-like layer (lower bracket). b-c. Staining forvarious brain region identities: forebrain (b); prefrontal cortex (notethe discrete boundary, arrow), Auts2 (c); hippocampus, Nrp2, Fzd9,Prox1. d. Hematoxylin-eosin staining of retinal tissue exhibitingstereotypical layering: retinal pigment epithelium (RPE), outer nuclearlayer (ONL) and inner nuclear layer (INL). Scale bars: 100 μm.

FIG. 11. Stereotypical organization and behavior of progenitors. a.Staining for the preplate marker Tbr1 (red) and neuronal marker MAP2(green) revealing superficial preplate (upper bracket) and underlyingneuronal IZ-like layer (lower bracket). b. Staining for the IP markerTbr2 (red) revealing SVZ localization of IPs (arrows).

FIG. 12. Organization and maturation of cerebral cortical neurons. a.Immunohistochemical staining at day 30 showing preplate (Tbr1) withearly signs of radial organization (MAP2, bracket i) and the presence ofan IZ-like layer (bracket ii) adjacent to the VZ/SVZ (bracket iii). DAPImarks nuclei (blue). b. Reelin staining indicating Cajal-Retzius cellsalong the basal surface of dorsal cortical tissue. c. Single celltracings of calcium surges with glutamate application (regions ofinterest, ROI, outlined in left panel) as measured by change influorescence (arbitrary units). Arrows mark the time of addition ofglutamate. d. Single cell tracing (ROIs marked in image at left) ofcalcium surges before (left panels) and after the addition of TTX (rightpanels). Scale bars: 100 μm.

FIG. 13. Cerebral organoid modeling of microcephaly.

a. Staining at day 22 showing increased neurons (Tuj1, arrows) inpatient-derived tissue (14B). b. BrdU pulse-chase in control andpatient-derived organoids (14B) showing higher percentage of BrdU cellswith neural identity and less in the VZ compared with control. Resultsquantified at right. Error bars are S.D. **P<0.01, Student's t-test. n=3organoids for each condition (300 cells total for control, 204 cells forpatient). c. P-Vimentin staining in control and patient-derived tissues(14B) showing RG mitotic divisions. Control RGs at anaphase dividedexclusively horizontal (0-30 degree angle, arrow) whereas patient RGsdisplayed many oblique and vertical orientations (arrowhead). Resultsquantified at right (P<0.01, 2×3 Fisher's exact test, n=11 cells forcontrol, n=15 cells for patient-derived, from >5 cortical regions each).

FIG. 14. Generation of cerebral organoids from multiple humanpluripotent stem cells. a. Hemotoxylin-eosin staining of cerebralorganoids compared with stationary culture reveals overall largertissues with substructure reminiscent of brain regions such as forebraincortex (arrows) and choroid plexus (arrowhead). b. Higher magnificationimages of hemotoxylin-eosin stained organoids revealing layeringreminiscent of the cerebral cortical molecular layer (bar), as well astissue reminiscent of meninges (arrowheads) and choroid plexus (arrows).c. TUNEL staining (green) revealing cell death in the interior regions(arrows) of the cerebral organoid with cortical regions developing alongthe exterior. DAPI marks nuclei (blue)

FIG. 15. Neural identity during differentiation of cerebral organoids.a. Staining for the cortical lobe markers Lmo4 (frontal and occipitalmarker, green) and Tshz2 (occipital marker, red). Note the expectednuclear staining (arrows, arrowheads) for both in one region (upperpanels) suggesting occipital identity, while only Lmo4 staining(arrowheads) is clearly evident in another region (lower panels)suggesting frontal identity. DAPI marks nuclei (blue). b. Staining forthe ventral marker Nkx2.1 (red) and the cortical interneuron markerCalretinin (green) on an organoid containing both ventral (arrowheads)and dorsal (upper left) regions within one section. Images at right arehigher magnification stitched images of the region outlined in the lowermagnification image at left. Calretinin interneurons can be seen betweenthe two regions with typical morphology of migration and redirectiontoward the dorsal cortex (arrows). Scale bars: 100 μm.

FIG. 16. Radial glial organization and morphology. a. Staining for thechromatin remodeling BAF components Baf53a (green, upper panels) andBaf53b (green, lower panels) in serial sections of the same tissueshowing the neural progenitor-specific Baf53a expressed in VZ RGs whilethe neuron-specific Baf53b is expressed in DCX+ (red) neurons outsidethe VZ. b. Higher magnification image of phospho-Vimentin staining(green) of a dividing radial glia revealing the long basal processtypical of radial glial morphology.

FIG. 17. Spatial organization and characteristics of cortical neuronidentities. a. Staining for the preplate marker Tbr1 (green) and thedeep-layer marker Ctip2 (red) at day 30 revealing rudimentary spatialseparation reminiscent of the early stages of CP development. b. Singlecell tracings of calcium surges in individual neurons (regions ofinterest, ROI, outlined in left panel) as measured by change influorescence (arbitrary units).

FIG. 18. Human features of cortical development not recapitulated inmouse organoids. a. Low magnification image of the region shown in FIG.5 a revealing the presence of a separated region of oRGs (demarcated byarrowheads) that appear separate from the VZ in all regions (brackets)but more separated and with a layer of Tuj1+ fibers in between inthicker parts of the cortical tissue (larger bracket). The entireorganoid can be seen in FIG. 1 c. b. Low magnification image of acerebral organoid derived from mouse ESCs stained for neurons (Tuj1,green) and neural progenitors (Sox2, red) revealing overall smallerorganoid size as well as smaller cortical regions (arrows) than human.c. Higher magnification of a region of cortical identity in mousecerebral organoids stained for RG progenitors (Sox2, red) revealing thepresence of only a few oRGs (arrowheads) that do not organize into aseparate layer such as that seen in human.

FIG. 19. Patient growth parameters. a. All growth parameters weresignificantly reduced both at birth and postnatally, with all z-scoresless than −2 standard deviations from the population mean for age andsex (dashed line). Weight (wgt), height (hgt) and head circumference(occipitofrontal circumference, ofc) at birth and at current age of 3½years of age. Head circumference was much more severely affected thanheight and weight, indicating that brain volume was disproportionatelyreduced as a result of more severe growth restriction.

FIG. 20. Characterization of patient derived iPSCs and cerebralorganoids. a. Quantification of the percentage of Sox2+ progenitors andTuj1+ neurons in cerebral cortical regions of control and 2 lines ofpatient derived tissues (1M and 14B) at the early stage of day 22. Errorbars are S.E.M. ***P<0.001 compared with control, Student's t-test. n=4tissues for each line. b. Bright-field image of patient-derived tissues(line 14B) electroporated with either GFP alone (left panel) or GFP andCDK5RAP2 expression construct (right panel). Note the presence of largerneuroepithelial tissue (arrows) in CDK5RAP2 electroporated tissuecompared with control. c. GFP staining (green) in GFP control (leftpanel) and CDK5RAP2 coelectroporated patient-derived tissues (14B)revealing the presence of multiple GFP+ neurons (arrowheads) in control6 days after electroporation, whereas CDK5RAP2 electroporated tissuesdisplay multiple GFP+ radial glia (arrows).

FIG. 21. shRNA mediated knockdown of CDK5RAP2 in human organoids. a.Western blot for endogenous CDK5RAP2 in 293T cells transfected with 4different shRNAs against CDK5RAP2. shRNA1 and 2 are most efficient whileshRNA 4 leads to a modest reduction in protein. Alpha-Tubulin is shownas a loading control. b. Quantification of percentage of GFP+electroporated cells exhibiting Sox2+ progenitor identity or DCX+neuronal identity in scrambled control or shRNA coelectroporatedtissues. ***P<0.001 compared to control, Student's t-test, n=4 tissuesfor each shRNA. Error bars are S.E.M.

EXAMPLES Example 1 Methods Plasmid Constructs and Materials

GFP plasmid used for coelectroporation with shRNA and for live imagingwas pCAG-GFP (Addgene plasmid 11150). shRNAs targeting human CDK5RAP2were cloned using pSuper shRNA expression strategy (OligoEngine).Targeting sequences were as follows: shRNA 1 AGGACGTGTTGCTTCAGAAAT (SEQID NO: 1), shRNA 2 AGAGTCAGCCTTCTGCTAAAG (SEQ ID NO: 2), shRNA 3GTGGAAGATCTCCTAACTAAA (SEQ ID NO: 3), shRNA 4 ACTATGAGACTGCTCTATCAG (SEQID NO: 4). The CDK5RAP2 expression construct was generated using theGateway system (Invitrogen) by PCR amplification of CDK5RAP2 from MGChuman CDK5RAP2 cDNA (clone ID: 9052276) using the primers with AttBsites: Forward: GGGGACAAGTTTGTACAAAAAAGCAGGCTTCATGATGGACTTGGTGTTGGAAGA(SEQ ID NO: 5), Reverse:GGGGACCACTTTGTACAAGAAAGCTGGGTCAGCTTTATTGGCTGAAAGTTCTTCTC (SEQ ID NO: 6).CDK5RAP2 was cloned into destination vector pcDNA3.1/nV5-DEST.

Cerebral Organoid Culture Conditions

Human H9 ES (WA09) were obtained from WiCell at passage 26 with verifiednormal karyotype and contamination-free. iPS cells were obtained fromSystem Biosciences (SC101A-1) verified pluripotent and contaminationfree. All human PSC lines were regularly checked and confirmed negativefor mycoplasma. Human embryonic stem (ES) or induced pluripotent stem(iPS) cells were maintained on CF-1 gamma irradiated MEFs according toWiCell protocols. On day 0 of organoid culture, ESCs or iPSCs weredissociated from MEFs by dispase treatment and MEFs were removed bygravity separation of stem cell colonies from MEFs before trypsinizationof stem cells to generate single cells. 4500 cells were then plated ineach well of an ultra-low binding 96-well plate in hES media with lowbFGF (5-fold reduced) and 50 uM ROCK inhibitor.

Embryoid bodies (EBs) were fed every other day for 6 days thentransferred to low adhesion 24-well plates in neural induction mediacontaining DMEM/F12, 1:100 N2 supplement (Invitrogen), Glutamax(Invitrogen), MEM-NEAA, and 1 ug/ml Heparin (Sigma). These began formingneuroepithelial tissues, which were fed every other day for 5 days. OnDay 11 of the protocol, tissues were transferred to droplets of Matrigelby pipetting into cold Matrigel on a sheet of Parafilm with small 3 mmdimples. These droplets were allowed to gel at 37 C and weresubsequently removed from the Parafilm and grown in differentiationmedia containing a 1:1 mixture of DMEM/F12 and Neurobasal containing1:200 N2 supplement, 1:100 B27 supplement without vitamin A(Invitrogen), 3.5 ul/L 2-mercaptoethanol, 1:4000 insulin (Sigma), 1:100Glutamax (Invitrogen), 1:200 MEM-NEAA.

After 4 days of stationary growth, the tissue droplets were transferredto a spinning bioreactor containing differentiation media as aboveexcept B27 supplement with vitamin A was used. Since retinoic acid hasbeen shown to be important for neuronal differentiation in vivo, weincluded it in the final media used to differentiate the cerebralorganoids.

Mouse Organoid Culture Conditions

Mouse A9 ES cells were cultured on Mitomycin C growth inactivated MEFsand passaged according to standard protocols (Tremml et al. 2008). Forthe generation of mouse organoids, the organoid protocol was appliedwith the following modifications: cells were trypsinized and 2000 stemcells were plated in each well of an ultra-low binding 96-well plate indifferentiation medium as described by Eiraku et al. (medium containing10 uM SB431542 but without Dkk-1). Subsequent steps were followedaccording to the human organoid method using identical mediacompositions, with the exception that for mouse tissues faster timingwas used according to morphology. EBs were transferred to neuralinduction medium on day 4, embedded in matrigel droplets on day 6, andon day 9 transferred to the spinning bioreactor.

Organoid Electroporation

Electroporation was performed using a petri dish tissue electrode andelectro-square-porator (ECM 830) both from BTX Harvard Apparatus. Atotal of 3 ul of 2 ug/ul total plasmid (GFP for live imaging, 1.8 ug/ulshRNA+0.2 ug/ul GFP for shRNA experiments) was injected in 4-5 locationswithin the organoid and electroporation was performed in differentiationmedia without antibiotics at 5 pulses, 80V, 50 ms duration, 1 secinterval. For rescue experiments, GFP expression plasmid and theCDK5RAP2 construct were coelectroporated at equal concentrations (1ug/ul each).

Live Imaging in Organoids

Live imaging was performed using a LSM780 confocal laser scanning system(Zeiss) equipped with temperature and CO₂ control. For calcium imaging,Fluo-4 direct (Life Technologies) was prepared according to manufacturerand applied 60 min before the start of imaging. Imaging was performed at494 nm excitation and 516 nm emission, frames taken every 20 sec for 100frames. Data analysis of calcium imaging was performed using ImageJ(Fiji). Regions of interest (ROIs) were manually selected and meanfluorescence was calculated for each time frame. Change is fluorescencewas calculated as follows: ΔF/F=(F−F_(basal)))/F_(background) whereF_(basal) was the lowest mean fluorescence value across imaging whileF_(background) was the average mean fluorescence across all frames.Glutamate was added by bath application to media during imaging at afinal concentration 100 uM. TTX was added by bath application to mediaduring imaging at a final concentration of 1 uM and imaging was resumedafter a 10 min incubation time.

Histology and Immunofluorescence

Tissues were fixed in 4% paraformaldehyde for 20 min at 4° C. followedby washing in PBS 3 times 10 min. Tissues were allowed to sink in 30%sucrose overnight and then embedded in 10%/7.5% gelatin/sucrose andcryosectioning at 20 μm. Tissue sections were stained withhemotoxylin/eosin or used for immunostaining. For immunohistochemistry,section were blocked and permeabilized in 0.25% Triton-X, 4% normaldonkey serum in PBS. Sections were then incubated with primaryantibodies in 0.1% Triton-X, 4% normal donkey serum at the followingdilutions: N-Cadherin (mouse, BD Biosciences 610920, 1:500), Sox2(rabbit, Chemicon, AB5603, 1:300), Tuj1 (mouse, Covance MMS-435P,1:750), TUNEL (In Situ Cell Death Detection Kit-Fluorescein, Roche),FoxG1 (rabbit, Abcam ab18259, 1:200), Emx1 (rabbit, Sigma HPA006421,1:50), Krox20 (rabbit, Covance PRB-236P, 1:100), Pax2 (mouse, AbnovaH00005076-M01, 1:200), Lmo4 (goat, Santa Cruz sc-11122, 1:50), Tshz2(rabbit, Sigma SAB4500379, 1:50), Otx1+2 (rabbit, Abcam ab21990, 1:200),Gbx2 (goat, Santa Cruz sc22230, 1:100), Auts2 (rabbit, Sigma HPA000390,1:250), Nkx2.1 (rabbit, Epitomics 6594-1, 1:250), Pax6 (mousemonoclonal, DSHB, 1:200), Pax6 (rabbit, Covance PRB-278P, 1:300),Calretinin (mouse, Swant 6B3, 1:100), Nrp2 (goat, RandD systems AF2215,1:40), Fzd9 (rabbit, Acris SP4153P, 1:200), Prox1 (mouse, ChemiconMAB5654, 1:200), TTR (sheep, AbD Serotec AHP1837, 1:100), Tbr2 (rabbit,Chemicon AB9618, 1:500), Tbr1 (rabbit, Abcam ab31940, 1:300), MAP2(mouse, 1:300), PH3 (rabbit, Cell Signaling Technology 9706S, 1:300),P-Vimentin (mouse, MBL International D076-35, 1:250), BrdU(preincubation in 2N HCl 20 min 37 C, rat, AbD Serotec OBT0030CX,1:500), Baf53a (rabbit, Bethyl IHC-00287, 1:250), Baf53b (rabbit, Abcamab140642, 1:250), Reelin, (mouse, Millipore MAB5366, 1:200), Ctip2 (rat,Abcam ab18465, 1:100), Satb2 (rabbit, Abcam ab34735, 1:100), DCX (goat,Santa Cruz sc-8066, 1:300), Brn2 (goat, Santa Cruz sc-6029, 1:40).Secondary antibodies used were donkey AlexaFluor 488, 568, and 647conjugates (Invitrogen, 1:500). For sections stained for BrdU, sectionswere first incubated with 2N HCl at 37° C. for 20 min followed bywashing three times in PBS before blocking.

RT-PCR

Total mRNA samples were isolated from whole organoids or hES cells intriplicate using Trizol reagent (Invitrogen). Potential contaminatingDNA was removed using DNA-Free (Ambion) and 1 ug RNA was used for cDNAsynthesis using Super-Script III (Life Technologies). PCR conditions andnumber of cycles (25-35 cycles) for each primer pair were empiricallydetermined using hES cDNA or human fetal brain cDNA (Invitrogen). Cycleswere run at 94° C. denaturation for 30 sec, 58-62° C. annealing for 45sec, depending on primer pair, and 72° C. extension for 30 sec. Primerpairs used were as follows: Oct4a For ggagaagctggagcaaaacc (SEQ ID NO:7), Rev tggctgaataccttcccaaa (SEQ ID NO: 8); Nanog Forgatttgtgggcctgaagaaa (SEQ ID NO: 9), Rev ctttgggactggtggaagaa (SEQ IDNO: 10); Sox1 For tatcttctgctccggctgtt (SEQ ID NO: 11), Revgggtcttcccttcctcctc (SEQ ID NO: 12); Pax6 For agttcttcgcaacctggcta (SEQID NO: 13), Rev attctctccccctccttcct (SEQ ID NO: 14); Actb Foraaatctggcaccacaccttc (SEQ ID NO: 15), Rev agaggcgtacagggatagca (SEQ IDNO: 16); BF1 For aggagggcgagaagaagaac (SEQ ID NO: 17), Revtgaactcgtagatgccgttg (SEQ ID NO: 18); Six3 For ctatcaacaacccccaacca (SEQID NO: 19), Rev agccgtgcttgtcctagaaa (SEQ ID NO: 20); Krox20 Forttgaccagatgaacggagtg (SEQ ID NO: 21), Rev cttgcccatgtaagtgaaggt (SEQ IDNO: 22); Isl1 For gctttgttagggatgggaaa (SEQ ID NO: 23), Revactcgatgtgatacaccttgga (SEQ ID NO: 24).

Cell Culture and Western Blot

HEK293T cells were grown in 10% FBS/DMEM and split at 40% into a 6-welldish (BD Falcon) followed by transfection the next day using TurboFect(Thermo Scientific) with 5 ug plasmid DNA. Cells were lysed 2 days laterand western blot was performed using rabbit anti-CDK5RAP2 (A300-554A,Bethyl labs, 1:10,000) followed by blotting for mouse anti-alpha tubulin(mouse, Sigma T6199, 1:10,000). Dermal fibroblasts were obtained by skinpunch biopsy and were cultured in amnioMAX C-100 complete medium(Invitrogen) and maintained in a 37° C. incubator with 5% CO₂ and 3% O₂.Cells were lysed in 50 mM Tris-HCl pH 8, 280 mM NaCl, 0.5% NP₄0, 0.2 mMEDTA, 0.2 mM EGTA, 10% Glycerol supplemented with protease inhibitortablet (Roche). Protein samples were run on a 3-8% Tris-acetate gel(Invitrogen) followed by immunoblotting using rabbit anti-CDK5RAP2(A300-554A, Bethyl labs, 1:2,000) and mouse anti-vinculin (V9264, Sigma,1:2,000). To perform immunofluorescence, patient fibroblasts were fixedin −20° C. methanol for 7 min and then blocked in PBS/1% bovine serumalbumin. Cells were then incubated in rabbit anti-CDK5RAP2 (A300-554A,Bethyl labs, 1:2,000) and mouse anti-CPAP (SC-81432, Santa CruzBiotechnology, 1:100) in blocking solution. Secondary antibodies usedwere donkey AlexaFluor 488 and 568 conjugates (Invitrogen, 1:500).

Research Subject and Gene Identification

Genomic DNA was extracted from peripheral blood of Patient 3842 and thepatient's parents by standard methods. Informed consent was obtainedfrom the family and the study approved by the Multicentre ResearchEthics Committee for Scotland (04:MRE00/19). Whole exome capture andsequencing was performed at the Welcome Trust Sanger Institute (WTSI),UK. DNA was sheared to 150 bp lengths by sonification (Covaris, Woburn,Mass., USA) prior to whole exome capture and amplification using theSureSelect Human All Exon 50 Mb kit (Agilent, Santa Clara, Calif.).Fragments were sequenced using the Illumina Hiseq platform. 76 bp pairedend sequence reads were aligned to the UCSC genome browser hg19reference sequence using BWA. Sequence variants were obtained usingGenomeAnalysisTK (www.broadinstitute.org/gatk/) and annotated withtranscript and protein consequence, polyphen, condel and SIFT scores.Mutations were confirmed by bi-directional sequencing of PCR productsusing dye terminator chemistry on an ABI 3730 capillary sequencer(Applied Biosystems).

Patient iPSC Reprogramming

Patient skin fibroblasts were reprogrammed using lentiviral delivery ofOct4, Sox2, Klf4, and c-Myc. Lentivirus production: A DNA mix consistingof virus packaging vectors (tat, rev, gag/pol, 1.5 ug each, and vsv-g, 3ug) and the loxP flanked OKSM reprogramming vector (oct-4, klf4, sox2,c-myc, 30 ug) were transfected into 293 cells. In brief, 112.5 μlFugene6 was added dropwise to 2 ml DMEM under constant vortexingfollowed by a 10 min incubation at RT. The DNA mix was added to theDMEM/Fugene6 mix while vortexing to generate the final transfection mix.After a 15 min incubation at RT, the transfection mix was added onto 80%confluent 293 cells, cultured in 13 ml 293 culture medium.Virus-containing medium was harvested and replaced with fresh medium 48h, 60 h and 72 h after transfection. The viral supernatant was stored at4° C. Reprogramming of human dermal fibroblasts: 1×10⁵ dermalfibroblasts were seeded the day before infection onto 10 cm and 6 cm0.1% Gelatin-coated culture dishes. Cells were incubated for 12 h withviral supernatant 1:1 mixed with dermal fibroblast medium supplementedwith 4 μg/ml polybrene. Thereafter, cells were washed with 1×PBS andcultured for 2 more days in dermal fibroblast medium. After 2 daysmedium was switched to human iPSCs medium supplemented with 10 ng/mlbFGF (peprotech, cat.nr: 100-18B), 10 μM CHIR99021 (stemgent, cat.nr:04-0004) and 1 μM PD 0325901 (stemgent, cat.nr: 04-0006) and cellscultured for 21 days. Medium was changed every day. Outgrowing colonies,identified by morphological appearance, were picked and passaged oninactivated CF-1 MEFs (global stem, cat.nr: GSC-6201M). Patient derivediPS lines were compared to control iPS cells obtained from a healthydonor (System Biosciences, SC101A-1). Alkaline phosphatase staining wasperformed using Vector Blue Alkaline Phosphatase Substrate Kit (VectorLaboratories, SK5300). Quantifications in patient and control iPSCderived organoids were performed blinded using coded file names inImageJ.

Patient Clinical Synopsis

Patient A3842 exhibited growth restriction from fetal life, with markedreduction in brain size evident at 22/40 weeks gestation. Pregnancyprogressed otherwise normally and the patient was born at term weighing1.82 kg (−3.9 s.d.). Postnatally, growth was also reduced such thatheight at 3 years 7 months was 73 cm (−6.7 s.d.), and head circumference35 cm (−13.2 s.d.), in keeping with a severe disproportionatemicrocephaly. The patient had quite prominent eyes and conical shapedwide-space teeth, but was otherwise unremarkable on examination. Noneurological deficits or malformations in other systems were evident,aside from a mixed conductive/sensorineural hearing loss. Developmentmilestones were mildly/moderately delayed. Neuroimaging at 22/40gestation demonstrated a smooth brain (the Sylvian fissure normallyevident at this gestation was not present) with small frontal lobes andpartial absence of the corpus callosum. Postnatally, MRI demonstratedmicrocephaly with a simplified gyral pattern and a cerebral cortex ofnormal thickness. In summary, clinical findings were in keeping withprevious cases of CDK5RAP2 primary microcephaly (deafness has beenpreviously reported with CDK5RAP2), with growth parameters falling onthe primary microcephaly-microcephalic primordial dwarfism spectrumreported for other centrosomal microcephaly genes such as CENPJ andCEP152.

Example 2 The Spinning Droplet Method for Production of CerebralOrganoids

Recent progress with in vitro models of various organ systems hasdemonstrated the enormous self-organizing capacity for pluripotent stemcells to form whole tissues. In developing an approach to model thecomplexity and heterogeneity of the human brain, we built upon thisconcept and left out any patterning growth factors that wouldartificially drive particular brain regions. We focused instead onimproving upon the growth requirements of the tissue and providing theenvironment necessary for intrinsic cues to influence development ratherthan driving formation of specific brain regions extrinsically.

We began with a modified approach to generate neuroectoderm fromembryoid bodies similar to that used to generate neural rosettes (Xiaand Zhang. 2009). However, the key difference in our approach is thatthese neuroectodermal tissues were then maintained in 3D culture andembedded in droplets of Matrigel, which were then transferred to aspinning bioreactor to enhance nutrient absorption and allow for growthof larger more complex tissues (FIG. 1 a).

This spinning droplet approach led to the formation of large, continuousneuroepithelia surrounding a fluid filled cavity reminiscent of aventricle (FIG. 1 b). These neuroepithelia displayed characteristicexpression of the neural specific N-cadherin, which localizedspecifically to the inner surface reflecting apical-basal polaritytypical for developing neuroepithelium. Furthermore, the neuroepitheliumwas larger and more continuous than tissues generated similar to Eirakuet al. (2008), which instead formed an aggregate of several smallrosette-like neuroepithelia (FIG. 1 b, e).

When these tissues were allowed to continue to develop further,organoids formed very large (up to 4 mm in diameter), highly complexheterogeneous tissues with structural characteristics reminiscent ofvarious brain regions (FIG. 1 c-e), which could survive indefinitely(currently up to 10 months) when maintained in a spinning bioreactor.Histological and gross morphological analysis revealed regionsreminiscent of cerebral cortex, choroid plexus, retina, and meninges.Importantly, tissues typically reached a size limit likely due to thelack of a circulatory system and limitations in oxygen and nutrientexchange. Consistent with this, extensive cell death was visible in thecore of these tissues (FIG. 14 c), whereas the various brain regionsdeveloped along the exterior. Furthermore, cerebral organoids could bereproducibly generated with similar overall morphology and complexityfrom both human ES cells and induced pluripotent stem cells (iPSCs)(FIG. 7 a, b), suggesting this approach could be applied to a variety ofhuman pluripotent stem cells.

Example 3 Cerebral Organoids Display Various Discrete Brain Regions

Since gross morphological analyses suggested the cerebral organoidsdisplayed heterogeneous brain regions, we next sought to characterizeregion identity of these tissues. We first performed RT-PCR for severalmarkers of pluripotency and neural identity (FIG. 8) and found thatwhile the pluripotency markers Oct4 and Nanog diminished during thecourse of organoid differentiation, the neural identity markers Sox1 andPax6 were upregulated, indicating successful neural induction of thesetissues.

We next examined regional markers of neural identity in whole organoids(FIG. 2 a), which revealed the presence of both forebrain markers (BF1and Six3) as well as hindbrain (Krox20 and Isl1) markers suggesting aheterogeneous population within the tissue. However, we noticed that astissues developed to more advanced stages, forebrain markers remainedhighly expressed while hindbrain markers began to decrease, suggestingthe relative amounts within the tissues of these identities changed overthe course of differentiation. This is particularly interesting in lightof the fact that normal human brain development reflects a similarchange in relative amounts of these identities due to the developmentalexpansion of forebrain tissue, eventually constituting approximately 85%of the human brain.

We then examined whether cells with these brain region identitiesdeveloped as discrete regions within the organoids, as gross morphologywould suggest, or were randomly interspersed within the tissue. To testthis, we performed immunohistochemical staining for markers of forebrainand midbrain as well as hindbrain identities at two time points duringthe early development of these tissues (FIG. 2 b). We could clearlyidentify several regions of forebrain identity by Pax6 expression and offorebrain/midbrain identity, as determined by Otx1/2 expression. Theseregions were located adjacent to regions lacking these markers butpositive for hindbrain markers Gbx2, Krox20, and Pax2, which wasreminiscent of the early mid-hindbrain boundary, suggesting similarregional communication and likely mutual repression. We additionallyobserved that regions of Gbx2 positivity decreased in abundance asdevelopment progressed, similar to results seen in FIG. 2 a, whereasOtx1/2 positive forebrain tissues continued to expand.

We next examined further developed tissues to test whether subregions ofthe forebrain could be distinguished. We performed staining for theforebrain marker FoxG1 (FIG. 2 c), which labeled regions displayingtypical cerebral cortical morphology. Many of these regions were alsopositive for Emx1 (FIG. 2 d), indicating dorsal cortical identity. Wecould identify several discrete regions within the cerebral organoidsthat stained positively for this marker and displayed typical dorsalcortical morphology. We also tested for subspecification within thedorsal cortex, namely the frontal cortex, by staining for the markerAuts2 (FIG. 2 d). Auts2 staining could be seen in neurons labelingdistinct regions of dorsal cortex, suggesting subspecification ofcortical lobes within the tissues. Tshz2, a marker of the occipital lobe(FIG. 15 a), and Lmo4, a marker of frontal and occipital lobes butabsent in parietal (FIG. 15 b). These markers could be seen in neuronslabeling distinct regions of dorsal cortex, suggesting subspecificationof cortical lobes.

Furthermore, staining for other cerebral cortical regions, namely theventral cortex (FIG. 2 e) and hippocampus (FIG. 2 f), similarly revealeddiscrete regions within organoids that displayed these identities aswell. Strikingly, interneurons produced in ventral forebrain regionsexhibited a morphology and location consistent with migration fromventral to dorsal tissues (FIG. 15 b). Within dorsal cortex, theseneurons displayed neurites parallel to the apical surface, reminiscentof the migratory extensions seen in tangential migration in vivo (FIG. 5g). Notably, Calretinin positive interneurons were absent from dorsalcortex of organoids lacking a ventral region (4/4 Nkx2.1 negativeorganoids), suggesting interneurons originate in ventral forebrain tomigrate to the dorsal cortex. This suggests distant regions caninfluence one another in developing cerebral organoids.

Finally, other brain structures separate from these cerebral corticalidentities could be observed, namely choroid plexus (FIG. 2 g) and evenimmature retina (FIG. 10 d). Overall, all tissues examined displayedregions with dorsal cortical morphology (35/35, 100%), most displayedchoroid plexus (25/35, 71%) and several displayed ventral forebrainidentity as determined by Nkx2.1 immunoreactivity (12/35, 34%), whereasonly a few displayed retinal tissue (determined by presence of retinalpigmented epithelium, 4/35, 11%). These results suggest that cerebralorganoids developed a variety of brain region identities organized intodiscrete, though interdependent, domains.

Example 4 Dorsal Cortical Organization and Radial Glial Behavior isRecapitulated in Cerebral Organoids

Since we were interested in modeling development and disease of thehuman dorsal cortex, we next examined the organization of dorsalcortical regions within cerebral organoids. Staining for markers ofradial glial progenitors (RGs) and newborn neurons (FIG. 3 a) revealedtypical progenitor zone organization with RGs forming a layer adjacentto a large fluid-filled cavity reminiscent of a ventricle, suggestingthe formation of a ventricular zone (VZ). Staining for Tbr1 (FIG. 11 a)revealed proper development of neural identity and radial migration tothe developing preplate (precursor to CP). Furthermore, staining forneural progenitor and neural specific BAF components revealed thecharacteristic switch in chromatin remodeling complexes during neuralfate specification (FIG. 16 a). Furthermore, staining for theintermediate progenitor (IP) marker Tbr2 (FIG. 3 b) revealed a thinlayer of IPs adjacent to the VZ, which was reminiscent of thesubventricular zone (SVZ). Thus, dorsal cortical tissues display typicalprogenitor zone organization much like that seen in vivo.

We next examined whether the behavior of these progenitors reflectedthat seen in the mammalian cerebral cortex. We examined proliferationwithin these tissues by staining for phospho-histone H3 (PH3) (FIG. 3 c)and observed the majority of cells dividing at the apical surface,adjacent to the fluid-filled cavity, likely marking the divisions ofRGs, which typically divide on the apical surface. We could additionallyobserve occasional divisions outside the VZ likely reflectingtransit-amplifying divisions of IPs and potentially divisions of arecently identified stem cell population, outer radial glia (discussedin more detail below).

Furthermore, when we stained for phospho-Vimentin (FIG. 3 d), a markerof mitotic RGs, we could observe the majority of divisions occurring atthe apical surface, similar to PH3 staining, but we could also observeclear basal processes extending all the way to the outer surface ofthese tissues (FIG. 3 e). This suggests RGs within these tissuesrecapitulated the typical apical-basal morphology seen in vivo.

To examine this in more detail, we sought to label individual RGs usingan electroporation approach. Drawing from our experience with in uteroelectroporation in the mouse embryonic brain, we developed a techniqueto inject plasmid DNA encoding GFP into the fluid filled cavities ofthese tissues and then apply a square-wave pulse electric field toelectroporate RGs adjacent to these ventricle-like cavities (FIG. 3 f).This approach led to reproducible expression of GFP within severalregions and in cells located adjacent to fluid-filled cavities.

When we examined GFP labeled cells within these dorsal cortical regions,we could identify RGs with typical morphology at various stages ofdevelopment (FIG. 3 g). For example, in earlier stage tissues, RGsdisplayed neuroepithelial morphology reflecting the pseudostratifiedstructure seen early in development. However, later stage tissuesdisplayed RGs with longer extended apical and basal processes reflectingthe bipolar morphology of these cells.

The observation that division of RGs occurred at the apical surface,suggested that RGs may undergo typical interkinetic nuclear migration.To test this, we performed live imaging of GFP electroporated RGs incerebral organoids. We could observe many examples of RGs that displayedmovement of the cell body along the apical and basal processes (FIG. 4a) consistent with interkinetic nuclear migration.

Furthermore, we performed pulse-chase experiments with the S-phasemarker BrdU to test whether nuclei of RGs shifted from outer VZlocalization towards the apical surface with time, as would be expectedif the cells were undergoing interkinetic nuclear migration. Indeed,following a short 1-hour pulse of BrdU, the majority of cells localizedto the outer region of the VZ (FIG. 4 b). However after washing and a4-hour or 6-hour chase we could observe progressively more cell nucleistained positively for BrdU closer to and adjacent to the apicalsurface. This is consistent with typical RG interkinetic nuclearmigration behavior.

We next examined the division mode of RGs at the apical surface. We hadalready observed that P-Vimentin stained mitotic RGs at the apicalsurface nicely (FIG. 4 c), and we could clearly discern the plane ofdivision from this staining. We therefore performed measurements of theplane of division (FIG. 4 d) to examine whether human RGs within thesecerebral organoids displayed similar mitotic orientations to those seenin other model systems, namely the developing mouse neocortex. Weobserved primarily planar orientations, which were parallel to theapical surface (FIG. 4 d), which has often been observed in developmentof other mammalian neocortex. However, we also observed quite abundantoblique orientations, which were present to a larger extent in thesehuman tissues than has typically been described for the developingrodent neocortex. Interestingly, these measurements reflected the sametrend recently described in the human brain, suggesting the cerebralorganoids could recapitulate aspects of human cortical development.

We further examined the fate potential of these divisions to testwhether RGs in human cerebral organoids could divide symmetrically orasymmetrically. We performed electroporation of GFP followed by a shortBrdU pulse-chase to lineage trace divisions of a small minority ofcells. When we examined double-labeled daughter cell pairs, we couldobserve both symmetric self-renewing RG fates, as well as asymmetricfates with only one daughter cell remaining an RG (FIG. 4 e, f). Thissuggests the RGs generated in these human tissues could undergo bothsymmetric and asymmetric divisions.

Example 5 Formation of Functional Cerebral Cortical Neurons

The formation of the radially organized CP begins with the formation ofits precursor, the preplate. To test for this initial organization, westained 30-day organoids for Tbr1, a marker of the preplate, as well asMap2, a neuronal marker38 (FIG. 12 a). This revealed the presence of abasal neural layer reminiscent of the preplate, and an apically adjacentregion reminiscent of the IZ. Furthermore, we could observe Reelinpositive neurons along the basal surface, suggesting the presence ofCajal-Retzius cells, an important population in generation of CParchitecture.

In vivo, dorsal cortical neurons mature and extend long-range axons. Totest for these characteristics, we performed GFP electroporation andexamined neuronal morphology. GFP-labeled axon projections displayedcomplex branching and growth cone behavior (FIG. 5 i) and projectedlong-range axons in a manner reminiscent of axon bundling (FIG. 5 h).

Finally, we tested whether neurons within cerebral organoids couldexhibited neural activity by performing calcium dye imaging to detectCa²⁺ oscillations, which revealed spontaneous calcium surges inindividual cells (FIG. 5 j, FIG. 17 b). Furthermore, we appliedexogenous glutamate (FIG. 12 c) and observed more frequent calciumspikes, indicating glutamatergic receptor activity. Finally, weperformed action potential blockade by application of tetrodotoxin (TTX)and observed dampened calcium surges indicating calcium spikes weredependent upon neuronal activity (FIG. 12 d).

Example 6 Recapitulation of Later Events in Human Cerebral CorticalDevelopment

In order to examine whether cerebral organoids could be used to studyhuman specific processes in neuronal development, we examined progenitorzone morphology in developmentally more advanced dorsal corticaltissues. These regions were typically much thicker and very large (asingle dorsal cortical region within an organoid could grow up to 1 mmacross) if allowed to develop to a more advanced stage. We stained forRGs and neurons and observed a large number of Sox2-positive progenitorsthat appear displaced from the apical surface (FIG. 5 a, FIG. 18 a). Themarker identity and location of these progenitors point to thepossibility that they represent outer radial glia (oRGs), a recentlyidentified progenitor type that is highly overrepresented in the humancerebral cortex compared with mice and other lower mammals.

To rule out the possibility that this OSVZ-like organization was an invitro artifact, we adapted the method to mouse ES cells to generatemouse cerebral organoids and examined whether a similar organization waspresent (FIGS. 18 b and c). We observed much smaller cortical tissues inmouse organoids compared with human, and only occasional oRGs that didnot accumulate in an OSVZ-like region. These results suggest OSVZ andIFL-like layers are specific to human organoids.

We furthermore observed that these fairly abundant oRGs appearedseparated from the apical VZ by a Tuj1 positive fiber layer (FIG. 5 a)reminiscent of the inner fiber layer seen in human but not mousedeveloping cortex. This organization suggests human cerebral organoidscould recapitulate at least some aspects of human-specific corticaldevelopment that cannot be modeled in mouse.

In order to further characterize these potential oRGs, we performedP-Vimentin staining to examine their morphology and observed obviousbasal processes emanating from these cells, whereas they lacked apicalprocesses (FIG. 5 b). This morphology, along with RG marker identity, isa hallmark of oRGs suggesting these basally displaced Sox2 andP-Vimentin positive progenitors indeed represent human oRGs.

We next examined the division mode of these oRGs and could identifyasymmetric divisions as labeled by daughter cell pairs with P-Vimentinin which only one daughter cell maintained Sox2 expression (FIG. 5 c).Furthermore, we could measure the division plane relative to the apicalsurface and found that the vast majority of oRGs divided perpendicularto the apical surface (FIG. 5 d). These findings suggest that cerebralorganoids could be a useful model system to study various aspects ofhuman oRGs.

As a final characterization of the human cerebral organoids, we soughtto describe the identity and behavior of the neurons produced in thedorsal cortical regions. We began by staining for cerebral corticallayer markers during advanced stages of development of these tissues.Previous methods of deriving cortical neurons have been able to generatevarious layer identity neurons, and we were similarly able to generateseveral layer identities using this approach. However, whereas othermethods have notably failed to recapitulate the spatial organization ofthe neuron layers, our cerebral organoids displayed at least rudimentaryseparation of layers (FIG. 5 e) and this spatial separation became morediscrete as tissues were allowed to develop (FIG. 5 f).

Furthermore, we observed an organization reminiscent of the inside-outpattern seen in developing mammalian cortex in vivo. Specifically, thelater born neurons marked by Brn2 and Satb2 localized more to the outerregions of the tissue while the earlier born neurons marked by Ctip2remained in the inner region (FIG. 5 e, f). This suggests these 3Dtissues may better recapitulate neuronal migration events than anypreviously described in vitro methods of generating cerebral corticalneurons.

Along these lines, we could even observe calretinin positive corticalinterneurons within the dorsal cortical plate and exhibiting migratoryprocesses parallel to the apical surface consistent with tangentialmigration (FIG. 5 g). Within other areas of these organoids, we couldidentify ventral cortical regions exhibiting calretinin positive neuronsquite removed from the dorsal cortex. This suggests the calretininpositive interneurons could migrate over a fairly long-range to reachtheir destination within the dorsal cortex, much like the developingcerebral cortex in vivo.

We next scrutinized the morphology of the dorsal cortical neurons byexamining GFP electroporated cells in tissues several days followingelectroporation. We could identify clusters of maturing corticalpyramidal cells, likely born at approximately the same time, thatprojected long-range axons together to the same distant location withinthe organoid (FIG. 5 h). Furthermore, pyramidal neuron axon projectionsdisplayed complex branching and growth cone behavior (FIG. 5 i) similarto that described in vivo.

Finally, we tested whether neurons produced within cerebral organoidsdisplayed neural activity by performing calcium imaging to detect Ca2+oscillations. Using the calcium sensitive dye Fluo-4, we could detectspontaneous calcium surges in individual neurons (FIG. 5 j). Thesefindings suggest cerebral organoid neurons were capable of maturationand synaptic activity.

Example 7 Cerebral Organoids Model Microcephaly and Implicate PrematureNeural Differentiation

Microcephaly is a neurodevelopmental disorder presenting with small(greater than 2 standard deviations below the mean) head circumference,which stems from the development of a greatly reduced brain size.Several genes have been identified in primary microcephaly as well asseveral overlapping disorders, such as microcephalic osteodysplasticprimordial dwarfism (MOPD) and Seckel syndrome. While evidence in modelsystems suggests many of the genes identified in these disorders mayfunction at the centrosome or in DNA repair, the human microcephalyphenotype has been notably difficult to model, as mouse mutants often donot display the same severity of phenotype. Since this disorder reflectsa defect in brain enlargement during development, and the human brainexhibits important divergences in mechanisms of expansion, wehypothesized that the human cerebral organoids may better model aspectsof this disorder.

We identified a patient with severe microcephaly (−13.2 standarddeviation below mean for age and sex) (FIG. 6 a) and reduced stature(−6.7 s.d.), who, as determined through exome sequencing and confirmedby capillary sequencing (FIG. 6 b), had compound heterozygous truncatingmutations in the coding sequence of the previously identified primarymicrocephaly gene CDK5RAP2 (FIG. 6 b). Both mutations led to prematurestop codons in a similar region of the protein, suggesting this mayreflect homozygous null mutation.

We obtained skin fibroblasts from this patient and performed westernblot (FIG. 6 c) as well as immunocytochemical staining for the Cdk5Rap2protein (FIG. 6 d). We could detect no protein in these patient cells,supporting the hypothesis that the microcephaly is due to the absence ofthe Cdk5Rap2 protein.

In order to model the phenotype in our organoid system, we nextperformed reprogramming of these patient skin fibroblasts usinglentiviral delivery of the four well-described reprogramming factors:Oct4, Sox2, c-Myc, and Klf4. We were able to generate severalindependent clones of iPSCs and characterized four of these formorphology and pluripotency. All four lines exhibited similar doublingtimes as well as colony morphology that were indistinguishable fromcontrol human iPSCs (FIG. 9 a). All lines could form embryoid bodies andexhibited positive staining for the pluripotency marker alkalinephosphatase (FIG. 9 b).

We next performed cerebral organoid culture from all of these 4 linesand could observe that when transferred to neural induction media, EBsfailed to develop further compared with control, and instead remainedquite small (FIG. 9 c). We hypothesized that since the patient alsodisplayed dwarfism, perhaps overall growth was perturbed as well. Wetherefore modified the protocol slightly by plating double the startingnumber of iPSCs thereby allowing EBs to develop further beforetransferring to neural induction. Indeed this approach allowed for theformation of neuroectoderm and subsequent neural tissue. However, grossmorphology revealed that all four lines displayed smallerneuroepithelial tissues and a large degree of neuronal outgrowthcompared with control tissues (FIG. 6 e and FIG. 9 d).

In order to examine this further, we allowed the tissues to an advancedstage and examined the overall morphology by immunohistochemicalstaining for progenitors and neurons (FIG. 6 f). We could observeoverall smaller neural tissues with only very few regions exhibitingprogenitors surrounding very small fluid-filled lumens compared withcontrol. These overall smaller neural tissues were reminiscent of thegreatly reduced brain size seen in humans with microcephaly.

We next sought to examine the cause of the hypoplasia seen in thesepatient cerebral organoids. To this end, we examined earlier stagetissues by immunohistochemistry for progenitors and neurons. Whereascontrol tissues at this stage displayed an abundance of largefluid-filled tissues primarily composed of progenitors, we could observeonly occasional small fluid-filled lumens surrounded by progenitors inthe patient derived tissues (FIG. 6 g, FIG. 20 a). Furthermore, patienttissues exhibited relatively increased neurons compared with controlsuggesting premature neural differentiation (FIG. 6 h), perhaps at theexpense of progenitors. To test this possibility, we performed BrdUpulse-chase experiments (FIG. 13 d) revealing a dramatic increase in thenumber of BrdU+/DCX+ cells in patient organoids, consistent withpremature neurogenic non-proliferative divisions.

Since these patient tissues lack the Cdk5Rap2 protein even beforeinitiation of neural induction, we next investigated whether an acuteloss of the protein after the formation of cerebral organoids would leadto a similar defect. To this end, we performed RNAi mediated knockdownof Cdk5Rap2 by co-electroplating GFP along with three independent shRNAs(shRNA1, shRNA2, shRNA4) found to knockdown endogenous Cdk5Rap2 in human293T cells (FIG. 9 e). All three shRNAs gave similar results, namely astriking loss of Sox2+ progenitors in the zone of electroporation and anincrease in DCX+ newborn neurons (FIG. 6 i). Of note, shRNA4 gave aweaker phenotype likely because this shRNA did not exhibit the sameefficiency of knock-down.

Finally, we tested whether the phenotype could be rescued byreintroducing CDK5RAP2 protein. We performed coelectroporation of GFPand CDK5RAP2 into day 12 patient organoids and examined 6 days later.Since high overexpression of CDK5RAP2 was toxic (data not shown), thecells with high GFP signal did not survive to this time point. However,we could observe regions in CDK5RAP2 electroporated tissues with largerneuroepithelium compared with tissues electroporated only with GFP(Extended Data FIG. 7 g). This effect could be due to surviving cellswith a low-level of CDK5RAP2 re-expression. Supporting thisinterpretation, staining for GFP (FIG. 20 c) revealed many low-levelGFP+ cells in CDK5RAP2 coelectroporated patient organoids with radialglial morphology (54%+/−2 SEM, n=74 cells from 3 tissues). In contrast,GFP+ cells in patient organoids electroporated with GFP alone exhibitedmainly neuronal morphology with significantly fewer radial glia(19%+/−11 SEM, n=102 cells from 3 tissues, P<0.05, Student's t-test).Thus, we conclude that the phenotype is specific to loss of CDK5RAP2.

When we examined this phenotype in more detail, we could observe thatvirtually all of the GFP shRNA co-electroporated cells exhibited neuralmorphology and costaining for DCX (FIG. 6 j). These findings suggestthat, similar to patient derived tissues, acute knockdown of Cdk5Rap2leads to premature neural differentiation at the expense of progenitors.This could lead to the overall size decrease seen in patient derivedtissues as well as patients with microcephaly since a loss ofprogenitors would be expected to lead to a final decrease in overalltissue growth.

As a further independent approach, we performed RNAi knockdown ofCDK5RAP2 by co-electroplating GFP with two independent shRNAs found toknockdown endogenous CDK5RAP2 (FIG. 21 a). Both shRNAs led to a strikingloss of Sox2⁺ progenitors and an increase in DCX⁺ neurons (FIG. 6 j,FIG. 21 b) reflecting a statistically significant increase in neuronproduction rather than progenitor maintenance (FIG. 21 b). Thesefindings support the conclusion that loss of CDK5RAP2 leads to prematureneural differentiation at the expense of progenitors.

Example 8 Recapitulation

Human brain development exhibits a number of unique characteristics thatwe are only beginning to tease out. Most of what we know about humanbrain development has been limited to fundamental processes shared withrodents and other lower mammals. While these insights have beenindispensible in understanding basic mechanisms of brain development,these neurodevelopmental studies have been limited by the model systemsavailable.

We have established a novel approach to studying humanneurodevelopmental processes through in vitro culture of cerebralorganoids from human pluripotent stem cells. This method recapitulatesnot only these basic mechanisms of neurodevelopment shared with mice andrats, but also displays many characteristics of human brain development.We are hopeful that this method will allow the study of a variety ofhuman specific neurodevelopmental processes.

Furthermore, a primary goal in neuroscience is to understand the rootsof human neurological disease. We have modeled at least some aspects ofthe human neurodevelopmental disorder microcephaly in these cerebralorganoids. The finding that progenitor zones in patient derived tissuesdisplay premature neural differentiation at the expense of earlyprogenitors supports a model in which the founder population of radialglial progenitors fails to properly expand in patient tissues, therebyleading to an overall smaller brain.

This may also explain why mouse models have been unable to recapitulatethe severity of the disorder in humans. It is hypothesized that themouse founder population of neural progenitors do not undergo expansionto the same extent as in human before the onset of neurogenesis. Thus, adisruption of this expansion in the founder population in mice would notlead to as severe of an effect as that seen in humans. Overall, ourfindings suggest we can utilize this in vitro culture system to modelaspects of human neurodevelopment and neurological disease and hopefullyprovide novel insight into the root causes of these disorders.

REFERENCES

-   Barrera et al. Dev Cell (2010) 18 (6): 913-26-   Bond et al. Nat Genet (2002) 32 (2): 316-20-   Bond et al. Nat Genet (2005) 37 (4): 353-5-   Cox et al. Trends Mol Med (2006) 12 (8): 358-66-   Eiraku et al. Cell Stem Cell (2008) 3: 519-532-   Elkabetz et al. Genes Dev (2008) 22 (2): 152-65-   Fietz et al. Nat Neurosci (2010) 13 (6): 690-9-   Fietz and Huttner. Curr Opin Neurobiol (2011) 21 (1): 23-35-   Götz and Huttner. Nat Rev Mol Cell Biol (2005) 6 (10): 777-88-   Hansen et al. Nature (2010) 464 (7288): 554-561-   Kenny et al. Mol Oncol (2007) 1 (1): 84-96-   Koch et al. Proc Natl Acad Sci USA (2009) 106 (9): 3225-30-   Lizarraga et al. Development (2010) 137 (11): 1907-17-   Lui et al. Cell (2011) 146 (1): 18-36-   Megraw et al. Trends Cell Biol (2011) 21 (8): 470-80-   Price and Brewer. Protocols for Neural Cell Culture: Third Edition.    (2001): 255-64-   Pulvers et al. Proc Natl Acad Sci USA (2010) 107 (38): 16595-600-   Reynolds and Weiss. Science (1992) 255 (5052): 1707-10-   Sato et al. Nature (2009) 459 (7244): 262-5-   Shi et al. Nat Neurosci (2012) 15 (3): 477-86, S1-   Thornton and Woods. Trends Genet (2009) 25 (11): 501-10-   Tremml et al. Curr Protoc Stem Cell Biol Chapter 1, Unit 1C.4 (2008)-   Wang et al., Nature Neuroscience, 14 (5) (2011): 555-561-   Wilson and Stice. Stem Cell Rev (2006) 2 (1): 67-77-   Xia and Zhang. Methods Mol Biol (2009) 549: 51-8-   Zhang et al. Nat Biotechnol (2001) 19 (12): 1129-33    These references are incorporated herein by reference. No mentioning    of references shall be construed as an acknowledgement of prior art.

1-36. (canceled)
 37. An in vitro grown artificial three-dimensionalneuronal tissue culture comprising a heterogeneous population of cellsof at least two different progenitor and neuronal differentiationlayers, wherein at least one progenitor layer comprises outer radialglia cell and the tissues further comprising an outer or extra corticalsubventricular zone and cells of a cortical inner fiber layer; and saidculture comprises a three dimensional matrix.
 38. An in vitro grownartificial three-dimensional neuronal tissue culture comprising aheterogeneous population of cells of at least two different progenitorand neuronal differentiation layers, wherein at least one progenitorlayer comprises outer radial glia cell and the tissues furthercomprising an outer or extra cortical subventricular zone and cells of acortical inner fiber layer; and said culture is obtained from culturinga neuronal differentiated multicellular aggregation in a threedimensional matrix.
 39. The tissue culture according to claim 37,wherein said tissue sections form at least two layers.
 40. The tissueculture according to claim 37, wherein at least one layer is shapedaround a globular tissue body.
 41. The tissue culture according to claim37, wherein said tissue develops apical and dorsal tissue sections. 42.The tissue culture according to claim 37, wherein said tissue iscerebral tissue.
 43. The tissue culture according to claim 37, whereincells of said culture express one or more gene expression markersselected from forebrain markers BF1 and Six3, hindbrain markers Krox20and Ils1.
 44. The tissue culture according to claim 37, whereinforebrain markers are expressed in increased amounts as compared tohindbrain markers.
 45. The tissue culture according to claim 37, whereincells of said culture express one or more gene expression markersselected from Otx1, Otx2, FoxG1, Auts2, Tuj1, Brn2, Satb2, Ctip2,calretinin.
 46. The tissue culture according to claim 37, wherein thethree dimensional matrix comprises a collagen.
 47. A method ofgenerating an artificial tissue culture comprising a) providing amulticellular aggregation of pluripotent stem cells, b) culturing saidmulticellular aggregation in neural induction medium thereby inducingthe multicellular aggregation to differentiate to neural tissue, c)culturing said differentiated multicellular aggregation in a threedimensional matrix, preferably a gel, thereby expanding said cells in amulticellular aggregation, wherein said cells are allowed todifferentiate further, and d) culturing said expanded multicellularaggregation of cells from step c) in a suspension culture.
 48. Themethod of claim 47, wherein said pluripotent cell is an inducedpluripotent cell, especially an induced pluripotent cell that has beenisolated from a patient.
 49. The method of claim 47, wherein saidexpanded cells differentiate into unipotent stem cells.
 50. The methodof claim 47, wherein the three dimensional matrix comprises collagen oran extracellular matrix from the Engelbreth-Holm-Swarm tumor, or anycomponent thereof selected from laminin, collagen, entactin, andheparan-sulfated proteoglycan or any combination thereof.
 51. The methodof claim 47, further comprising decreasing or increasing the expressionin a gene of interest in a cell at any stage during said method, whereinthe method is used for investigating a developmental neurological tissueeffect.
 52. The method of claim 47, further comprising administering acandidate agent to said cells at any stage during the method, preferablyat all stages, wherein the method is used for screening a candidatetherapeutic agent suitable for treating a developmental neurologicaltissue defect of interest.
 53. A method of testing a candidate drug forneurological effects, comprising administering a candidate drug to anartificial culture according to claim 37 and determining an activity ofinterest of the cells of said culture and comparing said activity to anactivity of cells to the culture without administering said candidatedrug, wherein a differential activity indicates a neurological effect.54. A method of obtaining a differentiated neural cell, the methodcomprising the step of providing an artificial culture according toclaim 37 and isolating a differentiated neural cell of interest.
 55. Amethod of obtaining a differentiated neural cell, the method comprisingthe step of generating an artificial tissue culture according to claim47 and isolating a differentiated neural cell of interest.
 56. A kitsuitable for generating an artificial three-dimensional neuronal tissueculture according to claim 1, the kit comprising at least: i) a mediumcomprising a three dimensional matrix and nutrients, and ii) a mediumcomprising retinoic acid and nutrients, and iii) a medium comprisingnutrients and one or more compounds selected from the group of a ROCKinhibitor, insulin and heparin.
 57. The kit of claim 56 furthercomprising iv) a further medium comprising nutrients but lacking growthfactors that would differentiate neural tissue to a particular fate.